The primary concern of ophthalmologists for age-related retinopathies and various sight-losing disorders is preserving the sight of patients. For example, open angle glaucoma, Leber's hereditary optic neuropathy, age-related macular degeneration, diabetic retinopathy, retinal ischemia, and a variety of retinitis pigmentosa. It has been known that various retinopathies and age-related ophthalmologic disorders lead to a progressive loss of vision.
Most often, these eye diseases are characterized by a gradual degeneration of retinal ganglion cells and eventual loss of the photoreceptors in the retina. (Quigley, H. A. 1999. Neuronal death in glaucoma. Prog. Retin. Eye Res. 18:39-57.) Cell death occurs through the process of apoptosis. Ischemic proliferative retinopathy (e.g., diabetes mellitus, retinopathy of prematurity, or retinal vein occlusion) is a major cause of blindness worldwide. Apart from neovascularization, ischemic proliferative retinopathy leads to retinal degeneration. Apoptosis has been ascribed to be the leading mechanism in ischemic retinal degeneration. (Adamis A P, Aiello L P, D'Amato R A (1999) Angiogenesis and ophthalmic disease. Angiogenesis 3:9-14). Apoptosis also is involved in age-related macular degeneration. (Hinton D M et al., Arch. Ophthalmol., 116:203-209 (1998). However, at the present time, there is no cure for these age-related eye diseases and blindness.
Glaucoma, an optic neuropathy affecting nearly 60 million people, is the second leading cause of blindness worldwide. (Quigley H A, Broman A T (2006) The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol 90:262-267). Visual field changes in glaucoma are believed to be caused by the loss of retinal ganglion cells (RGCs), although the exact cause of RGC degeneration is still unknown (Kuehn M H, Fingert J H, Kwon Y H (2007) Retinal ganglion cell death in glaucoma: mechanisms and neuroprotective strategies. Ophthalmol Clin North Am 18:383-395).
A novel gene, designated as puf-A, was cloned and functionally characterized, and its homologs in zebrafish, mouse, and human were identified as one of the three homolog clusters which were consisted of 14 related proteins with Puf repeats. Computer modeling of human Puf-A structure and a pull-down assay for interactions with RNA targets predicted that it was a RNA-binding protein. Knockdown of puf-A in zebrafish embryos resulted in microphthalmia, a small head, and abnormal primordial germ-cell migration. (Kuo M-W et al., A Novel puf-A Gene Predicted from Evolutionary Analysis Is Involved in the Development of Eyes and Primordial Germ-Cells. PLoS ONE 4(3): e4980. (2009)).
The Puf family is an evolutionarily conserved protein family named after Pumilio (Drosophila) and FBF (Fem-3 mRNA-binding Factor, Caenorhabditis elegans). Puf proteins have been found in various organisms, including yeast, C. elegans, Drosophila, zebrafish, Xenopus, mouse, and human, but their function is largely unclear. The first Puf protein, Pumilio, identified from Drosophila, was known to repress translation of hunchback mRNA in the posterior half of the Drosophila embryo, thereby permitting abdominal development. (Murata Y, Wharton R P (1995) Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos. Cell 80:747-756.) In addition to its role in posterior patterning of embryos, Drosophila Pumilio functions in the development of germline stem cells. (Braat A K, Zandbergen T, van de Water S, Goos H J, Zivkovic D (1999) Characterization of zebrafish primordial germ cells: morphology and early distribution of vasa RNA. Dev Dyn 216: 153-167). Puf family members are usually identified by the presence of eight tandem Puf repeats of ˜35-39 amino acids (Wang X, McLachlan J, Zamore P D, Hall T M (2002) Modular recognition of RNA by a human pumilio-homology domain. Cell 110: 501-512.) and the repeat binds to specific sequences in the 39 untranslated region (UTR) of a target mRNA.
The novel puf-A gene is involved in eye and primordial germ-cell (PGC) development. Analysis using the SMART server identified 14 puf-A related proteins in human, mouse and zebrafish. A computer modeling of human Puf-A predicted that it is a unique RNA-binding protein composed of six Puf repeats. (Kuo et al., PLoS ONE 4(3): e4980. (2009)). Computer modeling of human Puf-A structure and a pull-down assay for interactions with RNA targets predicted that it was a RNA-binding protein. Specifically, Puf-A contained a special six Puf-repeat domain, which constituted a unique superhelix half doughnut-shaped Puf domain with a topology similar to, but different from the conventional eight-repeat Pumilio domain. In mice, puf-A expression was detected primarily in retinal ganglion and pigmented cells of eye tissues. In some exemplary implementations is a unique puf-A gene that was shown to play a role in the development of eyes. The Puf-A protein, encoded by the unique puf-A gene, is expressed prominently in retinal ganglion cells. The expressed Puf-A protein has anti-apoptotic functions.
In zebrafish, the formation of retinal neurons follows a phylogenetically conserved order during embryogenesis. All six retinal neuron types are generated from common multipotent progenitors, with retinal ganglion cells being the first neurons to occur. Morphants of 3- and 5-days post fertilization (dpf embryos) have been shown to have incomplete differentiation patterns in the retina, thus indicating that the Puf-A protein plays a role in the development of retinal progenitors.
Additionally, during embryonic development, knockdown of the puf-A gene has led to a reduction in the number of PGCs and their abnormal migration. These results show that Puf-A is involved in the maintenance and migration of these primitive germ cells. The expression of puf-A was found predominantly in stage I follicles in adult ovaries and became undetectable in stage II and III follicles during subsequent oocyte development. It was noted that the most primitive germline stem cells, oogonia, were not readily distinguishable from stage I follicles. Thus, indicating that Puf-A not only regulates primordial germ cells (PGCs) development but may also play a role in germline stem cells up to stage I follicles. It is noteworthy that progressive ganglion cell degeneration precedes neuronal loss in many eye diseases.